1. Field of the Invention
The invention is in the field of pharmaceuticals, and provides drug conjugates as prodrugs for the delivery of drugs to cell populations, where the prodrugs are metabolized and activated by endogenous enzymes to provide active drugs.
2. Description of the Related Art
Metastatic carcinomas often express proteolytic enzymes including the cysteine protease cathepsin B (Demchik, L. L.; Sloane, B. F. Cell-Surface Proteases in Cancer. In Proteases. New Perspectives; A. Turk, Ed.; Birkhauser Verlag: Basel, 1999; pp 109–124; Mai, J.; Waisman, D. M.; Sloane, B. F. Cell Surface Complex of Cathepsin B/Annexin II Tetramer in Malignant Progression. Biochim. Biophys. Acta 2000, 1477, 215–230; Koblinski, J. E.; Ahram, M.; Sloane, B. F. Unraveling the Role of Proteases in Cancer. Clin. Chim. Acta 2000, 291, 113–135), matrix metalloproteinases such as collagenases and stromelysins (Davidson, A. H.; Drummond, A. H.; Galloway, W. A.; Whittaker, M. The Inhibition of Matrix Metalloproteinase Enzymes. Chem. Industry 1997, 258–261), and serine proteases, represented by plasminogen activator and plasmin (Andreasen, P. A.; Egelund, R.; Petersen, H. H. The Plasminogen Activation System in Tumor Growth, Invasion, and Metastasis. Cell. Mol. Life Sci. 2000, 57, 25–40). These enzymes are thought to be critically involved in the events that lead to metastasis, since they are capable of degrading the basement membranes and extracellular matrices around tumor tissues, allowing the tumor cells to migrate and invade into the surrounding stroma and endothelium. Additional activities associated with these proteases include participation in protease cascades, activation of enzymes and growth factors, and in tumor angiogenic stimulation.
Several investigators have explored the possibility of exploiting tumor-associated proteases for the development of new cancer chemotherapeutics. This has led to several promising orally active protease inhibitors having both preclinical and clinical antitumor activities (Davidson, A. H.; Drummond, A. H.; Galloway, W. A.; Whittaker, M. The Inhibition of Matrix Metalloproteinase Enzymes. Chem. Industry 1997, 258–261). An additional line of research involves the conscription of proteases for anticancer prodrug activation. Towards this end, peptide-containing anticancer prodrugs have been developed that are activated by proteases within solid tumors (Dubowchik, G. M.; Walker, M. A. Receptor-Mediated and Enzyme-Dependent Targeting of Cytotoxic anticancer Drugs. Pharm. Ther. 1999, 83, 67–123; Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A.; Weber, M. J. Protease-Activated “Prodrugs” for Cancer Chemotherapy. Proc. Natl. Acad Sci. USA 1980, 77, 2224–2228; Chakravarty, P. K.; Carl, P. L.; Weber, M. J.; Katzenellenbogen, J. A. Plasmin-Activated Prodrugs for Cancer Chemotherapy. 1. Synthesis and Biological Activity of Peptidylacivicin and Peptidylphenylenediamine Mustard. J. Med. Chem. 1983, 26, 633–638; Chakravarty, P. K.; Carl, P. L.; Weber, M. J.; Katzenellenbogen, J. A. Plasmin-Activated Prodrugs for Cancer Chemotherapy. 2. Synthesis and Biological Activity of Peptidyl Derivatives of Doxorubicin. J. Med. Chem. 1983, 26, 638–644; Dubowchik, G. M.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 1. A Model Study of Structural Requirements for Efficient Release of Doxorubicin. Bioorg. Med. Chem. Letts. 1998, 8, 3341–3346; Dubowchik, G. M.; Mosure, K.; Knipe, J. O.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 2. Models of Anticancer Drugs Paclitaxel (Taxol), Mitomycin C and Doxorubicin. Bioorg Med. Chem. Letts. 1998, 8, 3347–3352; de Groot, F. M. H.; de Bart, A. C. W.; Verheijen, J. H.; Scheeren, H. W. Synthesis and Biological Evaluation of Novel Prodrugs of Anthracyclines for Selective Activation by the Tumor-Associated Protease Plasmin. J. Med. Chem. 1999, 42, 5277–5283; de Groot, F. M. H.; van Berkon, L. W. A.; de Bart, A. C. W.; Scheeren, H. W. Synthesis and Biological Evaluation of 2′-Carbonate-Linked and 2′-Carbonate-Linked Prodrugs of Paclitaxel: Selective Activation by the Tumor-Associated Protease Plasmin. J. Med. Chem. 2000, 43, 3093–3102; Greenwald, R. B.; Pendri, A.; Conover, C. D.; Zhao, H.; Choe, Y. H.; Martinez, A.; Shum, K.; Guan, S. Drug Delivery Systems Employing 1,4- or 1,6-Elimination: Poly(ethylene glycol) Prodrugs of Amine-Containing Compounds. J. Med. Chem. 1999, 42, 3657–3667; Putnam, D. A.; Shiah, J. G.; Kopecek, J. Intracellularly Biorecognizable Derivatives of 5-Fluorouracil. Biochem. Pharm. 1996, 52, 957–962; Harada, M.; Sakakibara, H.; Yano, T; Suzuki, T.; Okuno, S. Determinants for the Drug Release from T-0128, Camptothecin Analogue-Carboxymethyl Detran Conjugate. J. Cont. Rel. 2000, 69, 399–412; Denmeade, S. R.; Nagy, A.; Gao, J.; Lilja, H.; Schally, A. V.; Isaacs, J. T. Enzymatic Activation of a Doxorubicin-Peptide Prodrug by Prostate-Specific Antigen. Cancer Res. 1998, 58, 2537–2540; Loadman, P. M.; Bibby, M. C.; Double, J. A.; Al-Shakhaa, W. M.; Duncan, R. Pharmacokinetics of PK1 and Doxorubicin in Experimental Colon Tumor Models With Differing Responses to PK1. Clin. Cancer Res. 1999, 5, 3682–3688). Several of these agents have led to significant in vitro and in vivo antitumor activities.
There are two general approaches for attaching drugs to peptides for intratumoral proteolytic activation. In the first approach, the drug is appended directly to the peptide, leading to prodrugs that can either release the parent drug or release a drug that contains vestiges of the bound peptide (Putnam, D. A.; Shiah, J. G.; Kopecek, J. Intracellularly Biorecognizable Derivatives of 5-Fluorouracil. Biochem. Pharm. 1996, 52, 957–962; Harada, M.; Sakakibara, H.; Yano, T; Suzuki, T.; Okuno, S. Determinants for the Drug Release from T-0128, Camptothecin Analogue-Carboxymethyl Detran Conjugate. J. Cont. Rel. 2000, 69, 399–412; Denmeade, S. R.; Nagy, A.; Gao, J.; Lilja, H.; Schally, A. V.; Isaacs, J. T. Enzymatic Activation of a Doxorubicin-Peptide Prodrug by Prostate-Specific Antigen. Cancer Res. 1998, 58, 2537–2540). In the latter case, the released drug may have impaired cytotoxic activity. An additional consideration for direct drug attachment to peptides is the negative influence the drug can have on the kinetics of peptide hydrolysis.
To circumvent these potential shortcomings, a second approach has been developed that relies on the use of self-immolative spacers to separate the drug from the site of enzymatic cleavage. The incorporated spacer allows for the release of fully active, chemically unmodified drug from the conjugate upon amide bond hydrolysis. A commonly used spacer utilizes the bifunctional p-aminobenzyl alcohol group, which is linked to the peptide through the amine moiety, thereby forming an amide bond. Amine-containing drugs are attached through carbamate functionalities to the benzylic hydroxyl group of the p-amionobenzyl alcohol-based spacer. The resulting prodrugs are activated upon protease-mediated cleavage, leading to a 1,6-elimination reaction (Wakselman, M. 1,4- and 1,6-Eliminations from Hydroxy- and Amino-Substituted Benzyl Systems: Chemical and Biochemical Applications. Nouveau J. Chim. 1983, 7, 439–447) that splits off unmodified drug and carbon dioxide.
This methodology, based on the work of Sartorelli, Katzenellenbogen and coworkers (Teicher, B. A.; Sartorelli, A. C. Nitrobenzyl Halides and Carbamates as Prototype Bioreductive Alkylating Agents. J. Med. Chem. 1980, 23, 955–960; Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A. A Novel Connector Linkage Applicable in Prodrug Design. J. Med. Chem. 1981, 24, 479–480) has been applied to plasmin catalyzed release of phenylenediamine mustard (Chakravarty, P. K.; Carl, P. L.; Weber, M. J.; Katzenellenbogen, J. A. Plasmin-Activated Prodrugs for Cancer Chemotherapy. 1. Synthesis and Biological Activity of Peptidylacivicin and Peptidylphenylenediamine Mustard. J. Med. Chem. 1983, 26, 633–638) and anthracyclines (Chakravarty, P. K.; Carl, P. L.: Weber, M. J.; Katzenellenbogen, J. A. Plasmin-Activated Prodrugs for Cancer Chemotherapy. 2. Synthesis and Biological Activity of Peptidyl Derivatives of Doxorubicin. J. Med. Chem. 1983, 26, 638–644; de Groot, F. M. H.; de Bart, A. C. W.; Verheijen, J. H.; Scheeren, H. W. Synthesis and Biological Evaluation of Novel Prodrugs of Anthracyclines for Selective Activation by the Tumor-Associated Protease Plasmin. J. Med. Chem. 1999, 42, 5277–5283; de Groot, F. M. H.; van Berkon, L. W. A.; de Bart, A. C. W.; Scheeren, H. W. Synthesis and Biological Evaluation of 2′-Carbonate-Linked and 2′-Carbonate-Linked Prodrugs of Paclitaxel: Selective Activation by the Tumor-Associated Protease Plasmin. J. Med. Chem. 2000, 43, 3093–3102) from their corresponding peptide-p-amidobenzyl carbamate derivatives, and also to release doxorubicin and mitomycin C from peptide-p-amidobenzyl carbamate peptide derivatives by lysosomal enzymes and cathepsin B (Dubowchik, G. M.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 1. A Model Study of Structural Requirements for Efficient Release of Doxorubicin. Bioorg. Med. Chem. Letts. 1998, 8, 3341–3346; Dubowchik, G. M.; Mosure, K.; Knipe, J. O.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 2. Models of Anticancer Drugs Paclitaxel (Taxol), Mitomycin C and Doxorubicin. Bioorg. Med. Chem. Letts. 1998, 8, 3347–3352; Greenwald, R. B.; Pendri, A.; Conover, C. D.; Zhao, H.; Choe, Y. H.; Martinez, A.; Shum, K.; Guan, S. Drug Delivery Systems Employing 1,4- or 1,6-Elimination: Poly(ethylene glycol) Prodrugs of Amine-Containing Compounds. J. Med. Chem. 1999, 42, 3657–3667). The same linkage system has also been applied for the activation of anthracyclines in cells that were transfected with carboxypeptidase G2 (Niculescu-Duvaz, I.; Niculescu-Duvaz, D.; Fiedlos, F.; Spooner, R.; Martin, J.; Marais, R.; Springer, C. J. Self-Immolative Anthracycline Prodrugs for Suicide Gene Therapy. J. Med. Chem. 1999, 42, 2485–2489).
The chemistry used for drug attachment has generally been restricted to amine-containing drugs, with the exception of paclitaxel, which was linked through carbonates formed from hydroxyl groups at the 2′ or 7-position (Dubowchik, G. M.; Mosure, K.; Knipe, J. O.; Firestone, R. A. Cathepsin B-Sensitive Dipeptide Prodrugs. 2. Models of Anticancer Drugs Paclitaxel (Taxol), Mitomycin C and Doxorubicin. Bioorg. Med. Chem. Letts. 1998, 8, 3347–3352; de Groot, F. M. H.; van Berkon, L. W. A.; de Bart, A. C. W.; Scheeren, H. W. Synthesis and Biological Evaluation of 2′-Carbonate-Linked and 2′-Carbonate-Linked Prodrugs of Paclitaxel: Selective Activation by the Tumor-Associated Protease Plasmin. J. Med. Chem. 2000, 43, 3093–3102). Unlike many carbonates that are hydrolytically unstable, these paclitaxel 2′ and 7-carbonates were quite stable in aqueous environments, consistent with what had already been reported for other paclitaxel carbonates (Ueda, Y; Matiskella, J. J.; Mikkilineni, A. B.; Farina, V.; Knipe, J. O.; Rose, W. C.; Casazza, A. M.; Vyas, D. M. Novel, Water-Soluble Phosphate Derivatives of 2′-Ethoxy Carbonylpaclitexel as Potential Prodrugs of Paclitaxel: Synthesis and Antitumor Evaluation Bioorg. Med. Chem. Letts. 1995, 5, 247–252; Senter, P. D.; Marquardt, H.; Thomas, B. A.; Hammock, B. D.; Frank, I. S.; Svensson, H. P. The Role of Rat Serum Carboxylesterase in the Activation of Paclitaxel and Camptothecin Prodrugs. Cancer Res. 1996, 56, 1471–1474). Many drugs containing reactive hydroxyl groups would not be expected to exhibit such high carbonate stability.
The present invention recognizes and addresses the need for broadly useful and versatile methodologies for attaching drugs, including anticancer drugs, to self-immolative spacers, which would lead to high serum stability and conditional drug release upon peptide bond hydrolysis.